The Central Role of Multiple P450 Genes and Their Co-factor CPR in the Development of Permethrin Resistance in the Mosquito Culex quinquefasciatus

Mosquitoes’ increasing resistance to insecticides is becoming a major threat for control efforts worldwide. Multiple P450 genes that are up-regulated in permethrin resistant strains of Culex quinquefasciatus have been linked to the development of resistance. In the current study, we characterized the function of six P450 genes, CYP6P14, CYP6BZ2, CYP9J33, CYP9J34, CYP9J40, and CYP9J45, that are overexpressed in the permethrin resistant Culex mosquitoes and showed their capability in metabolism of permethrin. These six P450 genes can convert 3-phenoxybenzoic alcohol (PBCHO) to a less toxic product, 3-phenoxybenzoic acid (PBCOOH), indicating that these P450s play an important role in permethrin degradation pathways. Although we know multiple P450 genes are over-expressed in permethrin resistant Culex mosquitoes, it remains to be seen whether cytochrome P450-reductase (CPR) gene that are co-overexpressed with P450 genes in permethrin resistant mosquitoes do indeed serve as a resistance mechanism. An in-depth investigation of the expression of CPR gene in resistant mosquitoes was conducted in permethrin resistant mosquitoes. The finding of CPR gene overexpression in permethrin resistant mosquitoes suggested the importance of co-overexpression of multiple P450 genes with their obligatory electron donor CPR in the complex detoxification system, boosting the metabolism of permethrin and hence the development of permethrin resistance in Cx. quinquefasciatus.

CANT-HYD: A Curated Database of Phylogeny-Derived Hidden Markov Models for Annotation of Marker Genes Involved in Hydrocarbon Degradation

Many pathways for hydrocarbon degradation have been discovered, yet there are no dedicated tools to identify and predict the hydrocarbon degradation potential of microbial genomes and metagenomes. Here we present the Calgary approach to ANnoTating HYDrocarbon degradation genes (CANT-HYD), a database of 37 HMMs of marker genes involved in anaerobic and aerobic degradation pathways of aliphatic and aromatic hydrocarbons. Using this database, we identify understudied or overlooked hydrocarbon degradation potential in many phyla. We also demonstrate its application in analyzing high-throughput sequence data by predicting hydrocarbon utilization in large metagenomic datasets from diverse environments. CANT-HYD is available at https://github.com/dgittins/CANT-HYD-HydrocarbonBiodegradation.

Bi/mZVI Combined with Citric Acid and Sodium Citrate to Mineralize Multiple Sulfa Antibiotics: Performance and Mechanism

The oxidative mineralization of sulfanilamide drugs (SAs) using micro-size zero-valent iron (mZVI) cooperated with a citric acid buffer solution was evaluated. In this study SM2, SMX, and SD could be removed at 66%, 89%, and 83%, respectively, in a 0.5% Bi/mZVI+CA+NaCA system within 2 h. Based on our analysis, the produced ·OH could be ascribed from the complexation between citrate iron (Fe(II)[Cit]−) and the generated H2O2 resulting from the activation of O2 on the mZVI surface in the Bi/mZVI+CA+NaCA system, further inducing the mineralization of antibiotics. The related possible degradation pathways were proposed. Two similar degradation pathways of SM2, SMX, and SD in the mixed liquid, including hydroxylation and SO2 extrusion, were solved. Meanwhile, there was an additional proposed degradation pathway for SMX to be degraded more effectively, as reflected in the opening of the N-O bond on the benzene ring. Therefore, this work provides an experimental basis and theoretical support for the efficient treatment of antibiotic wastewater in real industry by using an iron-based method.

Using Metabolite Data to Develop Patient Centric Specification for Amide Impurity in Vildagliptin Tablets

Many specified impurities in vildagliptin's finished product have been disclosed in the literature that are above their qualification threshold. We used the impurity B (amide impurity) as a case example to explore whether existing literature can be leveraged to determine the safe level of impurity and thereby develop a patient-centric specification (PCS) for impurities. No-observed-adverse-effect level (NOAEL) was derived from rate metabolism information and converted to human equivalent dose (HED). The HED was estimated as 6.5 mg/day. The high qualification levels are supported by repeat dose toxicity studies performed in rats, mice and dogs. Maximum theoretical amount (MTA) was correlated with the maximum observed amount (MOA) to verify whether the exposure was due to impurity and/or metabolite. MOA/MTA was found ≥1 suggesting that metabolism contributed to the amount excreted in feces and therefore could be used to further justify a higher specification limit than the usual one of ≤0.5%. Quite often the drug metabolism and degradation pathways overlap, resulting in the formation of identical constituents. Therefore, metabolism data can be leveraged for deriving safe levels of degradation impurities and develop PCS for impurities.